IFN receptor 1 binding proteins, DNA encoding them, and methods of modulating cellular response to interferons

ABSTRACT

Novel proteins IR1B1 and IR1B4 have been isolated which bind to the type I IFN receptor IFNAR1 and function in the cellular response to IFNs. DNA encoding such proteins in either the sense or anti-sense orientation can be administered to either enhance or inhibit the cellular response to IFNs. Antibodies to the proteins can be used for isolation of the new protein or for immunodetection thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.09/341,640, filed Oct. 8, 1999, now abandoned, which is the nationalstage under 35 U.S.C. 371 of PCT/US98/00671, filed Jan. 15, 1998, whichinternational application claims the benefit under 35 U.S.C. §119(e) ofU.S. provisional application No. 60/035,636, filed Jan. 15, 1997, nowabandoned.

FIELD OF THE INVENTION

The present invention relates generally to the molecular mechanisms ofinterferon action and, more specifically, to novel interferon receptor1-binding proteins, recombinant DNA molecules encoding them, and methodsfor modulating cellular response to interferon.

BACKGROUND OF THE INVENTION

Type I interferons (IFN-α and -β subtypes) produce pleiotropic effectson cells, such as inhibition of virus replication (antiviral effect),inhibition of cell proliferation (anti-tumoral effects), and modulationof immune cell activities (immunoregulatory effects). These multipleeffects of interferons (IFNs) are correlated with morphological andbiochemical modifications of cells (Revel, 1984, for review).

Interferons exert their activities through species-specific receptors.For type I IFNs, two transmembranal receptor chains have beenidentified: IFNAR1 (Uze et al, 1990) and IFNAR2-2 (or IFNAR2-c, Domanskiet al, 1995). Transduction of the signal generated by IFN-α, β, ωinvolves protein tyrosine kinases of the Janus kinases (Jak) family andtranscription factors of the Stat family (Darnell et al, 1994). Proteinsof the Jak-Stat pathways are activated by binding to theintracytoplasmic (IC) domains of the IFNAR1 and IFNAR2 receptor chains.Among the proteins binding to the IFNAR1 IC domain are tyk2 and Stat2(Abramovich et al, 1994). Stat2 would then recruit Stat1 to form theIFN-induced ISGF3 transcription complex which activates IFN-inducedgenes (Leung et al, 1995). Transcription complexes containing Stat3 arealso induced by IFN-β (Harroch et al, 1994) and an IFN-dependent bindingof Stat3 to IFNAR1-IC was observed (Yang et al, 1996). Protein-tyrosinephosphatase PTP1C and D reversibly associate with IFNAR1 upon IFNaddition (David et al, 1995a). In addition, two serine/threonine proteinkinases, the 48 kDa ERK2 MAP kinase (David et al, 1995b) and the cAMPactivated protein kinase A (David et al, 1996) bind to the isolatedmembrane-proximal 50 residues of IFNAR1-IC. Therefore, the type I IFNreceptor IC domains serve as docking sites for multiple proteins whichserve to generate and regulate the biological effects of IFNs on cells.

Two-hybrid screening in yeast is a potent method for identifying newproteins which bind to defined polypeptide sequences (Fields and Song,1989). Briefly, the two-hybrid screen is performed by transfecting yeastcells with (a) a plasmid DNA in which the defined polypeptide (bait) isfused to the DNA-binding domain of the Gal4 transcription factor, and(b) a cDNA library fused to the activation domain of Gal4 in a pACTplasmid. Yeast cells transfected with a cDNA that encodes for a proteinwhich binds to the polypeptide bait will then reconstitute the Gal4activity. The presence of such a protein which binds the polypeptidebait is revealed by expression of an enzymatic activity, such asβ-galactosidase, from a GAL1-lacZ construct that is preferablyintroduced into the yeast genome. From yeast clones which are positivein this test, it is possible to isolate the pACT plasmid, to determinethe nucleotide sequence of its insert and to identify the protein whichit encodes. This method has allowed the identification of novel proteinswhich interact with the IC domain of cytokine receptors (Boldin et al,1995).

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicants at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The present invention relates to two novel human proteins, hereindesignated IR1B1 and IR1B4, which have been identified to be IFNReceptor 1 (IFNAR1) binding proteins, and to the DNA encoding these twoproteins. Each of IR1B1 and IR1B4 proteins interacts with theintracytoplasmic (IC) domain of IFNAR1 and mediates the cellularresponses to interferon.

The present invention is directed to a recombinant DNA moleculecontaining a nucleotide sequence encoding either the IR1B1 or IR1B4proteins, or fragments thereof, as well as the proteins encoded thereby.In the recombinant DNA molecules, the nucleotide sequence encoding theIR1B1 or IR1B4 protein, or fragments thereof, is operably linked to apromoter in either a sense orientation or an anti-sense orientation.

By administering the recombinant DNA molecule containing a promoteroperably linked to the nucleotide sequence encoding a novel IFNAR1binding protein in the sense orientation directly into tumors, theresponse to exogenous interferon therapy in the treatment of cancer isenhanced.

Furthermore, the present invention also relates to a method ofprolonging tissue graft survival by introducing the recombinant moleculecontaining a promoter operably-linked to the nucleotide sequenceencoding a novel IFNAR1 binding protein, or fragment thereof, in theanti-sense orientation into the graft tissue prior to grafting to thepatient.

Thus, the present invention also relates to pharmaceutical compositionscontaining such DNA, RNA or protein and therapeutic methods for usingsame.

The present invention also relates to antibodies specific to the novelproteins of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the interaction of IR1B1 with the IFNAR1-IC domain asmeasured by the two-hybrid genetic interaction analysis in yeast. In theboxed lower portion of the figure, the cDNA insert in pACT as combinedwith various “baits” are indicated.

FIG. 2 shows the interaction of IR1B4 with the IFNAR1-IC domain asmeasured by the two-hybrid genetic interaction analysis in yeast. In theboxed lower portion of the figure, the cDNA insert in pACT as combinedwith various “baits” are indicated.

FIG. 3 shows the nucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO: 2)sequence of IR1E1.

FIG. 4 shows the homology and alignment of the amino acid sequence ofIR1B1 (SEQ ID NO:2) with the amino acid sequences of two calcium-bindingproteins, calcineurin B (abbreviated CALB; SEQ ID NO:3) and caltractin(abbreviated CATR; SEQ ID NO:4). Identical amino acids in IR1B1 and CALBor between CALB and CATR are shown by the symbol “|” therebetween.Identity between IR1B1 and CATR, but not with CALB, is shown by thesymbol “:” therebetween. Regions shown in bold type are the calciumbinding helix-loop-helix EF-hand domains.

FIG. 5 shows Northern blots of IR1B1 mRNA and 18S rRNA (lower line) inhuman myeloma U266S cells hybridized to IR1B1 cDNA and the rapid andtransient induction of IR1B1 upon treatment of the cells with eitherIFN-α8 or IFN-β for 2 hrs. or 18 hrs. The first line is a controlwithout IFN treatment after 2 hrs.

FIGS. 6A and 6B are SDS-PAGE lanes showing the in vitro interaction ofIR1B4 with the isolated IFNAR1-IC domain (FIG. 6A) and with cellextracts from human U266S and U266R cell membranes (FIG. 6B). In FIG.6A, the [³⁵S ]methionine-labeled translation products with or withoutflag-IR1B4 in vitro transcripts were either immunoprecipitated (10 μl)with anti-flag M2 beads (lanes 1 and 4), or reacted (50 μl) withglutathione beads coupled to GST fused to the 100 amino acid longIFNAR1-IC domain (lanes 2 and 5) or coupled to GST alone (lanes 3 and6). After overnight incubation at 4° C. (final volume 100 μl), the beadswere washed and SDS-eluted proteins boiled in reducing conditions beforeSDS-PAGE. In FIG. 6B, U266S (lane 1) or U266R cells (lane 2) wereextracted with Brij buffer and antiproteases (Abramovich et al, 1994)and 0.35 ml (10⁷ cells) was incubated with 75 μl of[³⁵S]methionine-labeled translation products of flag-IR1B4 transcriptsovernight at 4° C. Anti-IFNAR1 mAb R3 immobilized on protein G beads (25μl) was added for 2.5 hr, washed in Brij buffer, and SDS-eluted, boiledand reduced proteins analyzed by SDS-PAGE. A control with anti-flag M2beads as above was run (lane 3). The dried gels were visualized in aPhosphor-Imager. In the first three lanes of FIG. 6A, no IR1B4 mRNA wasadded to the in vitro translation reaction. In the second three lanes ofFIG. 6A, mRNA encoding IR1B4 protein fused to the flag protein wastranslated in an in vitro system.

FIG. 7 shows the nucleotide (SEQ ID NO:7) and deduced amino acidsequence (SEQ ID NO:8) of IR1B4.

FIG. 8 shows the amino acid alignment of IR1B4 (SEQ ID NO:8) and PRMT1(SEQ ID NO:9) and their differences.

FIG. 9 shows the amino acid alignment of IR1B4 and HCP-1 (SEQ ID NO:10)and their differences.

FIG. 10 shows a methyltransferase assay. Extract of U266S cells werereacted with beads coated with Protein A and anti-IFNAR1 antibody(lane 1) or with Protein A alone (lane 2). Methyltransferase activitywas measured by labeling of histones with ¹⁴C(methyl)-S-adenosylmethionine and analyzing radioactivity in the histone band byelectrophoresis on SDS-PAGE.

FIG. 11 shows an assay of protein-arginine methyltransferase activity inU266S cells. In lane 1, the protein-arginine methyltransferase activityof human U266S cells was measured by methylation of peptide R1, havingthe sequence of SEQ ID NO:11. In lane 2 an anti-sense oligonucleotide ofSEQ ID NO:12, complementary to the sequence of nucleotides 12–33 aroundthe initiation codon of IR1B4 cDNA, was added. In lane 3 thecorresponding sense oligonucleotide was added. It is seen that theanti-sense oligonucleotide substantially inhibits the protein-argininemethyltransferase activity while the control sense oligonucleotide haslittle effect.

FIG. 12 is a graph showing the growth inhibition of human U266S cells inresponse to IFN-β treatment in the presence or absence of the anti-senseoligonucleotide used in FIG. 11 (AS-1), the corresponding senseoligonucleotide (S-3), and another anti-sense oligonucleotide directedto the middle of IR1B4 cDNA (AS-2). Cell density was quantitated by acolor test with Alamar Blue (see Example 7) and the reduction in celldensity was calculated in percent of control wells untreated, andplotted as growth inhibition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery of two novel humanproteins which interact with the intracytoplasmic domain (IC) of theIFNAR1 chain of the interferon type 1 (IFN-α, β or ω) receptor and aredesignated herein as IFN Receptor Binding protein 1 (IR1B1) and IFNReceptor Binding Protein 4 (IR1B4). The interaction of these two novelproteins with IFNAR1 was demonstrated with a two-hybrid genetic test inyeast where transfection of the yeast reporter strain SFY526 (Bartel etal, 1993) with IR1B1 or IR1B4 cDNA fused to-the Gal4 activation domainresulted in β-galactosidase activity only when the IFNAR1-IC domain(fused to the Gal4 DNA-binding domain) was used as bait.

The sequence of IR1B1 cDNA encodes a 191 amino acid polypeptide.Computer searches of sequence databases revealed that IR1B1 is a novelprotein which shows marked homology, e.g., calcium binding sites (E-Fhandles), to the calcium binding proteins, calcineurin P and caltractin.Calcineurin P (Guerini et al, 1989) is a 19 kDa subunit of aserine/threonine phosphatase which plays a key role in activating thetranslocation of transcription factor NFAT to the nucleus ofT-lymphocytes, and which is inhibited by immunosuppressive drugs such ascyclosporin. Caltractin (Lee and Huang, 1993), a 21 kDa protein, is acytoskeleton-associated protein found in centrosomes, and is involved inthe movement of chromosomes during mitosis, and more generally inmicrotubule organization centers. Thus, the novel IR1B1 protein is a newmember of the calcineurin and caltractin family of calcium-regulatedproteins.

The gene for IR1B1 was surprisingly found to be rapidly activated inhuman cells by IFN treatment. Thus, this is the first example of acalcium-binding protein which is induced by IFN. Since calcium ionsregulate cell morphology, adhesion and division, modulation of IR1B1activity in cells could affect the response of normal and malignantcells to IFN. The role of IR1B1 in mediating the action of IFN in cellsis supported by the interaction of IR1B1 with the IC-domain of an IFNreceptor chain.

While IR1B4, like IR1B1, was found to be a novel protein as determinedby computer searches of sequence databases, it was also found that IR1B4has sequence homology to enzymes which utilize S-adenosyl methionine formethylating arginine residues in proteins and are designated as proteinarginine methyltransferases (PRMT1; Kagan and Clarke, 1994; Lin et al,1996). IR1B4 was found to bind directly to the IC-domain of IFNAR1 invitro, and the constitutive association of PRMT activity with the IFNARchain of the IFN-α, β receptor isolated from human cells wasdemonstrated by methylation of histones. When anti-senseoligodeoxynucleotides from the IR1B4 cDNA was added to human cellcultures, depletion of PRMT activity in the cell culture was observed.Human myeloma cells that were treated in this manner showed a muchreduced response to IFN as measured by growth-inhibition. Therefore,IR1B4/PRMT is involved in the pathway by which the IFN receptor causesgrowth-inhibition in tumor cells and is also involved in other functionsof the IFN receptor. Known substrates of PRMT include a number of RNAand DNA binding proteins, and in particular heterologous nuclearribonucleoproteins (hnRNPs). The hnRNPs are involved in mRNA transportfrom the nucleus to the cytoplasm, alternative splicing of pre-mRNA, andpost-transcriptional controls (Liu and Dreyfuss, 1995). Accordingly, thenovel human IR1B4/PRMT cDNA and protein, which were discovered by itsassociation with the IFN receptor, can be used to modify the response ofhuman or animal cells to IFN.

A recombinant DNA molecule according to the present invention contains anucleotide sequence that encodes the IR1B or IR1B4 protein, or afragment thereof, and can be used either to increase the cellularresponse to IFN by increasing expression of IR1B1 or IR1B4 cDNA or todecrease the cellular response to IFN by decreasing the expression ofIR1B1 or IR1B4 proteins with anti-sense RNA molecules.

The increased in vivo expression of IR1B1 or IR1B4 cDNA would be usefulin cancer therapy where the increased cellular response to IFN wouldresult in a decrease in malignant cell growth and an enhanced responseto exogenous IFN therapy. To obtain increased in vivo expression ofIR1B1 and IR1B4 at the target location for increased cellular responseto IFN, expression vectors containing IR1B1 or IR1B4 cDNAoperably-linked in a sense orientation to a strong constitutive promotercan be injected directly at the target location, such as into braintumors or metastatic tumor nodules (e.g., melanoma or breast cancer).

Conversely, the decreased in vivo expression of IR1B1 or IR1B4 proteinswould be useful in prolonging the survival of tissue grafts as therejection of these grafts in the host is mediated by thehistocompatibility antigens (MHC class I) whose synthesis depends on theIFN stimulus. When the cDNA of, IR1B1 or IR1B4, or fragments thereof,carried on a suitable vector and operably-linked in an anti-senseorientation to a promoter, is introduced into cells of the tissue to begrafted, the expression of anti-sense RNA leads to the degradation ofIR1B1 or IR1B4 mRNA (or sense RNA for IR1B1/IR1B4) and a consequentdecrease in the cellular levels of IR1B1 or IR1B4 protein.

Anti-sense RNA is transcribed from an upstream promoter operably-linkedto a coding sequence oriented in the anti-sense direction, i.e.,opposite the normal or sense direction of the DNA and its transcribedsense RNA (mRNA). The expression of anti-sense RNA complementary to thesense RNA is a powerful way of regulating the biological function of RNAmolecules. Through the formation of a stable duplex between sense RNAand anti-sense RNA, the normal or sense RNA transcript is renderedinactive and untranslatable.

Recombinant DNA molecules, as embodiments of the present invention,contain the cDNA of IR1B1 or IR1B4, or fragments thereof,operably-linked to a promoter in either a sense or anti-senseorientation. The term “promoter” is meant to comprehend adouble-stranded DNA or RNA sequence which is capable of binding RNApolymerase and promoting the transcription of an “operably linked”nucleic acid sequence. Thus, a DNA sequence would be operably linked toa promoter sequence if the promoter is capable of effecting thetranscription of the DNA sequence, regardless of the orientation of theDNA sequence.

The types of promoters used to control transcription may be any of thosewhich are functional in the host/target cells. Examples of promotersfunctional in mammalian cells include the SV40 early promoter,adenovirus major late promoter, herpes simplex (HSV) thymidine kinasepromoter, rous sarcoma (RSV) LTR promoter, human cytomegalovirus (CMV)immediate early promoter, mouse mammary tumor virus (MMTV) LTR promoter,interferon β promoter, heat shock protein 70 (hsp70) promoter, as wellas many others well known in the art.

A promoter operably linked to IR1B1 or IR1B4 cDNA in the senseorientation for expression of IR1B1 or IR1B4 protein is preferably astrong constitutive promoter. This allows for a high level of IR1B1 orIR1B4 regardless of the presence of endogenous cellular mechanisms forregulating the expression of IR1B1 or IR1B4.

Likewise, the promoter, which is operably linked to IR1B1 or IR1B4 cDNAin the anti-sense orientation, is preferably a strong promoter, such asthe promoter present in the Epstein-Barr Virus (EBV) regulating regionwhich allows for high levels of anti-sense RNA expression (Deiss andKimchi, 1991).

The anti-sense sequence is preferably only expressible in thehost/target cells, which are preferably human cells and the expressedanti-sense RNA should be stable (i.e., does not undergo rapiddegradation). The anti-sense RNA should only specifically hybridize tothe sense mRNA expressed in host/target cells, and form a stabledouble-stranded RNA molecule that is essentially non-translatable. Inother words, the anti-sense RNA expressed in host/target cells preventsthe expressed sense mRNA from being translated into IR1B1 or IR1B4proteins. The vector-borne anti-sense sequence may carry either theentire IR1B1 or IR1B4 cDNA sequence or merely a portion thereof, as longas the anti-sense portion is capable of hybridizing to sense mRNA andpreventing its translation into IR1B1 or IR1B4 protein. Accordingly, an“anti-sense” sequence as used throughout the specification and claims isdefined as the entire anti-sense sequence or a portion thereof which iscapable of being expressed in transformed/transfected cells, and whichis also capable of specifically hybridizing to “sense” IR1B1 or IR1B4mRNA to form a non-translatable double-stranded RNA molecule.

The anti-sense sequence need not hybridize to the entire length of theIR1B1 or IR1B4 mRNA. Instead, it may hybridize to selected regions, suchas the 5′-untranslated non-coding sequence, the coding sequence, or the3′-untranslated sequence of the “sense” mRNA. Preferably, the anti-sensesequence hybridizes to the 5′-coding sequence and/or 5′-non-codingregion, such as at cap and initiation codon sites, since it has beenobserved it has been observed with many examples of anti-senseoligonucleotides that targeting the initiation codon is more effective,whereas targeting internal sequences within the coding region is not aseffective (Wickstrom, 1991). The effectiveness of an anti-sense sequencein preventing translation of IR1B4 sense mRNA can easily be tested in anassay for protein-arginine methyltransferase activity in U266S cells asdescribed in Example 7. In view of the size of the mammalian genome, theanti-sense IR1B1 or IR1B4 sequence is preferably at least 17, morepreferably at least 30 base pairs in length. However, shorter sequencesmay still be useful, i.e., they either fortuitously do not hybridize toother mammalian sequences, or such “cross-hybridization” does notinterfere with the metabolism of the cell in a manner and to a degreewhich prevents the accomplishment of the objects of this invention.

Both the preferred hybridization target and the preferred anti-sensesequence length are readily determined by systematic experiment.Standard methods such as described in Ausubel et al, eds. CurrentProtocols in Molecular Biology, Greene Publishing Assoc., New York,N.Y., 1987–1996, and Sambrook et al, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989) can be used to systematically remove an increasingly largerportion of the anti-sense sequence from the vector. Besides the fulllength anti-sense sequence, a series of staggered deletions may begenerated, preferably at the 5′-end of the anti-sense sequence. Thiscreates a set of truncated anti-sense sequences that still remaincomplementary to preferably the 5′-end of the sense mRNA and as aresult, still forms an RNA molecule that is double-stranded at the5′-end of the sense mRNA (complements the 3′-end of an anti-sense RNA)and remains non-translatable. Moreover, anti-sense oligonucleotides,such as oligonucleotide AS-1 (SEQ ID NO:12), can be readily synthesizedchemically and introduced onto a vector in operable linkage with apromoter for use in decreasing the in vivo cellular expression of IR1B1or IR1B4 protein.

The vectors of the present invention may be any suitable eukaryotic orprokaryotic vector normally used for transfecting mammalian cells, suchas episomal, replicable, or chromosomally integratable vectorswell-known in the art. A particularly preferred vector for theexpression of IR1B1 or IR1B4 anti-sense RNA is the episomal plasmidcontaining the Epstein-Barr Virus regulatory region (Deiss and Kimchi,1991) to serve as the promoter that is operably-linked to IR1B1 or IR1B4cDNA arranged in an anti-sense orientation relative to this regulatoryregion. The use of anti-sense vectors and oligonucleotidephosphorothioates are addressed in Annals of the New York of Sciences:Gene Therapy for Neoplastic Diseases. eds. B. E. Huber and J. S. Lazo,Vol. 716, 1994 (e.g. Milligan et al, pp. 228–241).

According to the present invention, the survival of tissues or organsgrafted to a patient in need of such a graft can be prolonged bydecreasing the cellular response to IFN. Rejection of graft tissue ismediated by the histocompatibility antigens, with the synthesis of theseMHC class I antigens being dependent on IFN stimulus. Thus, a decreasein cellular response to IFN stimulus will prolong the survival of grafttissue. The method for prolonging tissue graft survival according to thepresent invention involves introducing into cells of a tissue or organto be grafted to a patient a recombinant DNA molecule containing a IR1B1or IR1B4 cDNA sequence, or fragment thereof, operably linked to apromoter in the anti-sense orientation, whereby anti-sense IR1B1 orIR1B4 RNA is expressed in such transfected/transformed cells. Therecombinant DNA molecule can be introduced into the cells of a tissue ororgan in any manner well-known in the art to be suitable for thispurpose. Following the introduction of the recombinant DNA molecule intocells of the tissue or organ, the tissue or organ can be grafted to thepatient in need of such a graft.

A pharmaceutical composition containing a recombinant DNA molecule,which is an expression vector and which carries IR1B1 or IR1B4 cDNAoperably linked to a promoter in a sense orientation, can be injecteddirectly into tumors, e.g., brain tumors and metastatic tumor nodules,to make the cells within these tumors more responsive to exogenous IFNtherapy as a treatment for cancer. The enhanced cellular response toexogenous IFN therapy would lead to an inhibition of malignant cellgrowth.

Gene transfer in vivo or ex vivo is well-reported, i.e., in Annals ofthe New York Academy of Sciences: Gene Therapy for Neoplastic Diseases,Vol. 716, 1994; see, for example, “Direct Gene Transfer for theUnderstanding and Treatment of Human Disease” by G. E. Plautz on pages144–153, and “Mechanisms of Action of the p53 Tumor suppressor andProspects for Cancer Gene Therapy by Reconstitution of p53 Function” byRoemer et al, on pages 265–282. Methods of inserting recombinant DNAmolecules into cells of a tissue or organ to be grafted or of a tumorinclude adenovirus, retrovirus, adenovirus-associated virus (AAV)vectors, as well as direct DNA injection or oligonucleotide-liposomeinjection. Clinical trials where retroviral vectors are injected intobrain tumors or where adenovirus is used to infect upper respiratorytract cells of a patient with cystic fibrosis are well-known.

Pharmaceutical compositions comprising the recombinant DNA moleculeencoding IR1B1 or IR1B4 cDNA, or a fragment thereof, either in a senseor anti-sense orientation with respect to an operably linked promoter,is intended to include all compositions where the recombinant DNAmolecule is contained in an amount effective for achieving its intendedpurpose. In addition, the pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers or excipients whichstabilize the recombinant DNA molecule or facilitate its administration.

Another embodiment of the present invention is directed to moleculeswhich include the antigen-binding portion of an antibody specific forIFNAR1-binding proteins IR1B1 or IR1B4, or fragments thereof, for use indiagnostics, such as immunodetection methods to assay for the level ofIR1B1 or IR1B4 proteins in tumor tissue obtained from biopsies or foruse in affinity chromatography purification of the protein.

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic(anti-Id) antibodies, single-chain antibodies, and recombinantlyproduced humanized antibodies, as well as active fractions thereofprovided by any known technique, such as, but not limited to enzymaticcleavage, peptide synthesis or recombinant techniques.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. MAbs may be obtained by methods known tothose skilled in the art. See, for example Kohler and Milstein, Nature256:495–497 (1975); U.S. Pat. No. 4,376,110; Ausubel et al, eds., supra,Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring HarborLaboratory (1988); and Colligan et al, eds., CURRENT PROTOCOLS INIMMUNOLOGY, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), the contents of which references are incorporated entirelyherein by reference. Such antibodies may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridomaproducing a mAb of the present invention may be cultivated in vitro, insitu or in vivo. Production of high titers of mAbs in vivo or in situmakes this the presently preferred method of production.

Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having the variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273–3277 (1984); Morrisonet al, Proc. Natl. Acad. Sci. USA 81:68516855 (1984); Boulianne et al,Nature 312:643–646 (1984); Cabilly et al, European Patent Application125023 (published Nov. 14, 1984); Neuberger et al, Nature 314:268–270(1985); Taniguchi et al, European Patent Application 171496 (publishedFeb. 19, 1985); Morrison et al, European Patent Application 173494(published Mar. 5, 1986); Neuberger et al, PCT Application WO 8601533,(published Mar. 13, 1986); Kudo et al, European Patent Application184187 (published Jun. 11, 1986); Morrison et al, European PatentApplication 173494 (published Mar. 5, 1986); Sahagan et al, J. Immunol.137:1066–1074 (1986); Robinson et al, International Patent Publication,WO 9702671 (published 7 May 1987); Liu et al, Proc. Natl. Acad. Sci. USA84:3439–3443 (1987); Sun et al, Proc. Natl. Acad. Sci. USA 84:214–218(1987); Better et al, Science 240:1041–1043 (1988); and Harlow and Lane,ANTIBODIES: A LABORATORY MANUAL, supra.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the mAb with the mAb to which an anti-Id is being prepared. Theimmunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See, for example,U.S. Pat. No. 4,699,880.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

It should be understood that antibodies of the present invention may beintact antibodies, such as monoclonal antibodies, but that it is theepitope binding site of the antibody which provides the desiredfunction. Thus, besides the intact antibody, proteolytic fragmentsthereof such as the Fab or F(ab′)2 fragments can be used. Fab andF(ab′)2 fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody (Wahl et al, J. Nucl. Med. 24:316–325(1983)). Such fragments are typically produced by proteolytic cleavage,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)2 fragments).

Furthermore, the DNA encoding the variable region of the antibody can beinserted into other antibodies to produce chimeric antibodies (see, forexample, U.S. Pat. No. 4,816,567) or into T-cell receptors to produceT-cells with the same broad specificity (see Eshhar, Z. et al, Br. J.Cancer Suppl., 10:27–9, 1990; Gross, G. et al, Proc. Natl. Acad. Sci.USA, 86:10024–8, 1989). Single chain antibodies can also be produced andused. Single chain antibodies can be single chain composite polypeptideshaving antigen binding capabilities and comprising a pair of amino acidsequences homologous or analogous to the variable regions of animmunoglobulin light and heavy chain (linked V_(H)–V_(L) or single chainFv). Both V_(H) and V_(L) may copy natural monoclonal antibody sequencesor one or both of the chains may comprise a CDR-FR construct of the typedescribed in U.S. Pat. No. 5,091,513. The separate polypeptidesanalogous to the variable regions of the light and heavy chains are heldtogether by a polypeptide linker. Methods of production of such singleantibodies, particularly where the DNA encoding the polypeptidestructures of the V_(H) and V_(L) chains are known, may be accomplishedin accordance with the methods described, for example, in U.S. Pat. No.4,946,778, 5,091,513 and 5,096,815.

Thus, the term “a molecule which includes the antigen-binding portion ofan antibody” is intended to include not only intact immunoglobulinmolecules of any isotype and generated by any animal cell line ormicroorganism, but also the reactive fraction thereof including, but notlimited to, the Fab fragment, the Fab′ fragment, the F(ab′)2 fragment,the variable portion of the heavy and/or light chains thereof, andchimeric or single-chain antibodies incorporating such reactivefraction, as well as any other type of molecule or cell in which suchantibody reactive fraction has been physically inserted, such as achimeric T-cell receptor or a T-cell having such a receptor, ormolecules developed to deliver therapeutic moieties by means of aportion of the molecule containing such a reactive fraction.

Having now fully described the invention, the same will be more readilyunderstood through reference to the following examples which areprovided by way of illustration and is not intended to be limiting ofthe present invention.

EXAMPLE 1 Two Human Proteins, IR1B1 and IR1B4, Bind to the IFN Receptor

A cDNA fragment encoding the entire IFNAR1-IC domain amplified by PCRusing a BamH1-sense primer (5′ctgaggatccAAAGTCTTCTTGAGATGCATC (SEQ IDNO:5)) and an EcoRI anti-sense primer (5′tgacgaattcctaTCATACAAAGTC (SEQID NO:6)), was cloned in a Bluescript vector (BS—SK⁺, Stratagene). TheBamHI-SalI fragment from this BS-IFNAR1-IC was introduced in the pGBT₁₀vector (CloneTech) and fused in-phase after the Gal4 DNA binding domain(pGBT₁₀-IFNAR1-IC) for two-hybrid screening. The two-hybrid screeningmethod (Fields and Song, 1989) was carried out with the modifiedprocedure of Durfee et al (1993) using the pACT plasmid cDNA libraryfrom human Epstein-Barr Virus (EBV)-transformed B-lymphocytes toco-transform yeast reporter strain Y153 with pGBT₁₀-IFNAR1-IC. The yeastY153 strain has two reporter genes under the control of GAL1 UpstreamActivating Sequences (UAS) which are transcribed only if the activity ofthe Gal4 transcription factor is reconstituted. This requires that thefusion protein encoded by the pACT plasmid which was introduced intothis particular yeast clone have affinity for the IFNAR1-IC domain fromthe pGBT10 plasmid. One of the reporter genes is GAL1 His3, which allowsfor growth in a medium lacking histidine; the other reporter gene isGAL-lacZ, which provides β-galactosidase activity. In addition, the pACTplasmids have the Leu2 gene and the pGBT₁₀ plasmid has the TRP1 genewhich allows for growth in a medium lacking leucine and tryptophan,respectively. Colonies were selected in synthetic medium SC minus Trp,Leu, His in the presence of 25 mM 3-aminotriazole (which further selectsfor histidine prototrophy). The growing colonies were then tested forβ-galactosidase activity using the X-gal filter assay (Breeden andNaysmith, 1985).

Nine positive yeast clones were obtained and their pACT plasmids wererecovered by transfection into E. coli HB101 and selection for leu⁺transformants. For each yeast DNA, two such E. coli HB101 clones wereisolated. Partial DNA sequencing of the pACT plasmids from these E. coliclones showed that they fell into two groups of cDNA sequences whichwere designated IR1B1 and IR1B4. The pACT plasmids of the IR1B1 andIR1B4 groups were subjected to specificity tests by co-transformation ofthe SFY526 yeast reporter strain (Bartel et al, 1993) with pAS plasmidsharboring lamin, cdk2 and p53 or other control inserts (CloneTech).Colonies which grew in SC -trp, -leu were tested for β-galactosidaseexpression. From the specifically positive pACT plasmids, inserts wereexcised with XhoI, cloned into BS—KS (Stratagene) and subjected tosequencing from T7 and T3 promoters using the DyeDeoxy Terminator CycleSequencing Kit in a 373A DNA Sequencer (Applied Biosystems).

FIG. 1 shows the results for pACT clone IR1B1 co-transfected into yeastSFY526 with different pAS or pGBT₁₀ plasmid baits. Yeast cells grew inthe selective SC medium -trp, -leu in streaks 1 to 9 of the filter.Staining by X-gal reagent(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was positive only instreaks 2 and 4. As indicated in FIG. 1, streak 4 is a control yeastwith an active lacZ gene. Streak 2 is the combination of IR1B1 andIFNAR1-IC fusion proteins. IR1B1 alone (streak 9), or any othercombination besides IR1B1 and IFNAR1-IC, did not exhibit β-galactosidaseactivity. Therefore, IR1B1 is specifically able to combine with the ICdomain of the IFNAR1 IFN receptor chain.

Similarly, FIG. 2 shows the results for pACT clone IR1B4 co-transfectedinto yeast SFY526 with different pAS or pGBT₁₀ plasmid baits. Yeastcells grew in SC medium trp, leu in streaks 1 to 8 of the filter andstaining by Xgal reagent was positive only in streaks 3 and 7. Asindicated in the lower boxed portion of FIG. 2, streak 7 is a controlyeast with an active lacZ gene. Streak 3 is the combination of IR1B4 andIFNAR1-IC fusion proteins. Like the results obtained with IR1B1, IR1B4alone (streak 1), or any other combination besides IR1B4 and IFNAR1-IC,did not exhibit β-galactosidase activity. Therefore, IR1B4 is alsospecifically able to combine with the IC domain of the IFNAR1 IFNreceptor chain.

EXAMPLE 2 IR1B1 Protein Sequence Shows Calcium-Binding EF Hand Sites

The cDNA insert of the pACT-IR1B1 plasmids was excised with restrictionenzyme XhoI, cloned into a Bluescript BS-KS vector and subjected tosequencing from T7 and T3 promoters using the DyeDeoxy Terminator CycleSequencing kit in a 373A DNA sequencer (Applied Biosystems). The longestplasmid had a sequence of 830 nucleotides (FIG. 3) following the Gal4Activation domain and linker sequence of the pACT plasmid and an openreading frame of 191 amino acids was found therein (FIG. 3). An onlinesearch of the protein databases was performed using the Blast algorithm(Altschul et al, 1990) as well as the Bioaccelerator Alignment (Henikoffand Henikoff, 1992). The highest scores were obtained for caltractin(CATR_HUMAN, accession Swiss Protein SW New P41208) with 62.1 1%similarity and 32.4% identity from amino acids 52 to 173, and forcalcineurin B (CALB NAEGR, accession Swiss Protein P42322; CALB_HUMAN,accession P06705) with 59.8% similarity and 32.5% identity from aminoacids 50 to 171.

FIG. 4 shows the alignment of IR1B1 with human calcineurin B (CALB) andcaltractin (CATR). The calcium binding, helix-loop-helix EF-hand domainsare shown in bold and underlined characters. IR1B1 has two EF-hand sitesbut the first two EF-hand domains are not conserved. IR1B1 showshomology to both calcineurin B (represented by vertical lines in FIG. 4)and caltractin (represented by colons in FIG. 4). However, IR1B1 isclearly a novel and different human protein which has not beenpreviously identified.

EXAMPLE 3 IR1B1 is an IFN-Induced Gene Product

Human myeloma U266S cells (about 3×10⁶ cells in 5 ml suspensioncultures) were treated with recombinant IFN-α8 (2×10⁸ IU/mg frombacteria) or with recombinant IFN-β (3×108 IU/mg from CHO cells) at 750IU/ml for 2 hours or for 18 hours. After treatment with IFN, the cellswere collected and extracted with Tri-reagent (Molecular ResearchCenter, Cincinnati, Ohio), which is a product containing guanidiniumthiocyanate and phenol. The extracted RNA was ethanol precipitated,denatured with formaldehyde, analyzed by electrophoresis informaldehyde-agarose gels (10 μg RNA/slot), and blotted on GeneScreenPlus (Dupont, New England Nuclear, Billerica, Mass.). The Northern blotwas reacted with 106 cpm of IR1B1 cDNA labeled with the Rediprime kit(Amersham, UK) using ³²P-dCTP and random priming.

FIG. 5 shows that the IR1B1 cDNA hybridized to a 1.1 kb RNA. The amountof IR1B1 mRNA was markedly increased 2 hours after IFN-β treatment ofU266S cells. However, at 18 hours after IFN treatment, the IR1B1 mRNAhad disappeared from the cells, indicating that the induction is bothrapid and transient. Many IFN-induced mRNAs continue to accumulate inthe cells for over 24 hours after IFN treatment (Revel and Chebath,1986).

It was verified that the same amount of RNA was present in each lane. Asshown on the lower part of FIG. 5, hybridization of the same U266S (richin IFN receptor) RNA to an 18S ribosomal cDNA probe reveals the sameamount of 18S rRNA in each lane (only the part of the blot where 18SrRNA runs is shown). In another experiment using 1,200 U/ml of IFN forinduction, IR1B1 mRNA was also observed with IFN-α8 at 2 hours, but notat 30 minutes (not shown).

The IR1B1 mRNA was found to have the same 1.1 kb size in different humancells (U266, Daudi and THP-1 cells). It is notable that this size isclose to that of caltractin mRNA but not to that of calcineurin B mRNA(2.5 kb). The small size of the mRNA is consistent with IR1B1 being asmall protein of about 20 kDa.

EXAMPLE 4 IR1B4 Protein Binds to IFNAR1 in Vitro

The binding of IR1B4 to the IC-domain of IFNAR1 was tested bysynthesizing the IR1B4 protein with a protein tag (flag sequence) usingin vitro translation in reticulocyte lysates and reacting this proteinwith a recombinant IFNAR1-IC fusion protein in E. coli. The pact-IR1B4DNA from Example 1, cut with XhoI and filled-in by Klenow enzyme, wascloned in the PECE-flag expression vector (Ellis et al, 1986) cut withEcoRI and filled-in. The NotI-BamHI fragment containing the in-frameflag-IR1B4 fusion was recloned in BS-SK cut with NotI-BamHI anddownstream from the T3 promoters. The sequence of the flag fusion wasverified by sequencing from the T3 promoter. In vitro transcription(Promega kit) was done with T3 polymerase and 1 μg of BamHI-linearizedBS-flag-IR1B4 DNA. In vitro translation was carried out in rabbitreticulocyte lysates (Promega kit) with [³⁵S]methionine (Amersham) and 5μg of RNA transcripts for 1 h. at 30° C. The products were RNase treatedbefore use. The GST-IFNAR1-IC fusion protein was prepared by cloning theBamHI-EcoRI, insert of BS-IFNAR1-IC (see above) into the same sites ofpGEX2 (Pharmacia Biotech). GST and GST-IFNAR1-IC were expressed in E.coli and recovered bound to Glutathione-Agarose beads (Sigma).

Anti-flag M2 agarose beads were from Kodak Scientific Imaging Systems.Monoclonal antibodies IFNaR3 to the α-component of the IFN receptor(IFNAR1) were a kind gift of Dr. O. Colamonici (Colamonici et al, 1990)and were used at 1:100 dilution. Rabbit antibodies to the C-terminalpeptide of IFNAR1-IC (Ab 631) were prepared and used forimmunoprecipitation of IFNAR1 from Brij extracts (0.75 ml) of 2×10⁷human myeloma U266S and U266R cells with antiproteases previouslydetailed (Abramovich et al, 1994) except that protein G beads(Pharmacia) were used with mAb IFNaR3 SDS-PAGE and analysis in a FujixBAS1000 Phosphor Imager were as before (Harroch et al, 1994).

It was first verified that a protein product of about 32 kDa is obtainedwhen the translation products were immunoprecipitated by anti-flagantibodies (FIGS. 6A and 6B). In FIGS. 6A and 6B, whenever the use ofanti-flag antibodies is noted (by+sign), it means that the radioactivetranslation product of the IR1B4-flag fusion mRNA (in vitro transcribedfrom the corresponding DNA construct) was reacted with anti-flag M2antibody bound to agarose beads (product of Kodak Scientific ImagingSystems). The translated protein which contains IR1B4 fused to the flagamino acid sequence was bound to these anti-flag antibody beads andafter centrifuging down the beads, the protein was eluted with SDSbuffer and applied onto SDS-PAGE. These reactions serve as a control todemonstrate that the expected fused protein is present.

Beads of Glutathione-Sepharose (Sigma), to which the GlutathioneS-transferase (GST) fused to IFNAR1-IC was bound, were added to thereticulocyte lysate translation reaction. The beads were centrifuged andwashed and the proteins bound to GST beads were released by sodiumdodecyl sulfate (SDS 1%) and analyzed by SDS-polyacrylamide gelelectrophoresis (PAGE). The 32 kDa protein labeled by ³⁵S-methionine wasobserved to be bound to GST-IFNAR1-IC but not to GST alone (FIG. 6A).This demonstrates that IR1B4 directly binds to the isolated IFNAR1-ICpeptide region.

To verify that IR1B4 interacts with the IFNAR1 protein as present inhuman cell membranes, detergent extracts of human myeloma U266 cellswere mixed with the ³⁵S-methionine labeled translation products of IR1B4mRNA from reticulocyte lysates. The IFNAR1 protein wasimmunoprecipitated by a monoclonal antibody IFNaR3 specific to theectodomain of IFNAR1 (from Colamonici et al, 1990). Analysis by SDS-PAGEshowed the presence of the 32 kDa IR1B4-flag band (FIG. 6B) when thedetergent extracts originated from U266S (rich in IFN receptor), but notwhen originating from U266R cells—a mutant IFN-α, β-resistant derivativecell line from U266 deficient in IFN receptors (Abramovich et al, 1994).The 32 kDa band similarly was seen when U266S extracts were reacted withAb 631 against the C-terminal peptide of IFNAR1, and IFNAR1 wasprecipitated by anti-flag when Cos-7 cells were transferred by flg-IR1B4and human IFNAR1 cDNAs. These results demonstrated that IR1B4 binds tointact IFNAR1 from human cells in a specific manner.

EXAMPLE 5 IR1B4 cDNA and Protein Sequences

The nucleotide sequence of the IR1B4 cDNA has an open reading frameencoding a 361 amino-acid long protein (FIG. 7). This human cDNArecognized a 1.5 kb constitutively expressed poly-A⁺ mRNA in varioushuman cells including U266 myeloma cells. An online search of theprotein databases was performed using the BlastP algorithm (Altschul etal, 1990) as well as the Bioaccelerator Alignment (Henikoff andHenikoff, 1992), and it was found that IR1B4 is a unique member of theprotein-arginine methyltransferase family. The rat PRMT1 cDNA describedby Lin et al (1996, Genbank sequence I.D. 1390024; Accession U60882) isonly 81.4% homologous when analyzed by the ALIGN computer program. Atthe amino acid level (FIG. 8), the human IR1B4/PRMT differs clearly inits amino terminus from PRMT1, with the first 19 amino acids beingcompletely different. N-terminal sequencing of IR1B4 alone would nothave provided any indication that IR1B4 is homologous to PRMT1. Anotherhuman protein which has been described, HCP-1 (Nikawa et al, 1996;Genbank accession D66904) was also found to have homology to IR1B4.However, HCP-1 has a different amino acid sequence from residues 147–175(FIG. 9). HCP-1 was originally identified based on its ability tocomplement the irel5 mutation in yeast and its enzymatic function wasnot previously identified (Nikawa et al, 1996). Therefore, IR1B4 is anovel human protein.

EXAMPLE 6 IR1B4 Protein Bound to IFNAR1-IC Has MethyltransferaseActivity

Methyltransferase activity could be co-immunoprecipitated from humancell extracts with the IFNAR1 receptor. Brij-detergent extracts of U266Scells were reacted overnight at 4° C. with or without anti-IFNAR1antibody Ab 631 (Abramovich et al, 1994). Protein A beads (40 μl of a50% of IPA-400 fast flow, Repligen) were added for 1 hour. The beadswere washed and incubated in 0.1 ml of 25 mM Tris-HCl, pH 7.5, 1 mMEDTA, 1 mM EGTA, 50 μM (0.25 μCi) ¹⁴C-(methyl)-S-adenosyl-methionine(Amersham), and 100 μg histones (Type IIA from calf thymus, Sigma) for30 min. at 30° C. The in vitro methylation of histones was carried outunder the conditions described by Lin et al (1996). The radioactivity inthe histone band was analyzed after SDS PAGE (15% acrylamide) andexposure in the Phosphor-imager. A ¹⁴C-methyl labeling of the histoneswas observed with the beads that were coated with anti-IFNAR1, but notwith those in the control reaction (FIG. 10). Therefore, proteinmethyl-transferase activity is constitutively associated with the IFNreceptor chain of these human cells. A similar enzyme activity wasrecovered when IFNAR1 was immunoprecipitated five minutes after additionof IFN-β to the U266S cells.

EXAMPLE 7 Involvement of IR1B4/PRMT1 in IFN Action

An antisense oligodeoxynucleotide phosphorothioate (Stein et al, 1989)complementary to the sequence of nucleotides 12–33 around the initiationcodon of IR1B4 cDNA (AS-1, anti-sense sequence5′GGCTACAAAATTCTCCATGATG-3′; SEQ ID NO:12) was synthesized chemically.The oligonucleotides were added to U266S cells seeded in 96-wellmicroplates (8000 cells/well/0.2 ml RPMI, 10% FCS) at a finalconcentration of 10 μM on day 0 and re-added at 5 μM on day 2. IFN-β wasadded at 64 or 125 IU/ml on day 0. After 3 days of culture, 20 μl ofAlamar Blue, a colorimetric cell density indicator based onoxido-reduction (BioSource, Camarillo, Calif.), was added to each welland incubation continued for 6–7 h. Color was measured in a microplateELISA reader (test filter 530 nm, reference filter 630 nm) with multiplereading of duplicate wells. Correlation of the growth curves by livecell number and by OD was verified. To measure methyltransferase, cellsfrom pooled wells were lysed by freeze-thawing in 25 μl/well of 25 mMTris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 40 μg/ml leupeptin andaprotinin, 20 μg/ml pepstatin, 1 UM phenylmethylsulfonyl fluoride(PMSF). Reactions were in 50 μl with 25 μl of cell extracts, 100 μMpeptide R1 (Najbauer et al, 1993; obtained from Genosys, Cambridge, UK),3 μCi of [³H] (methyl)S-adenosylmethionine (Amersham, 73 Ci/mmol) for 30min at 30° C. After electrophoresis in SDS-polyacrylamide (16%) gel,fixation in 50% methanol, 10% acetic acid and treatment by Amplify(Amersham), autoradiography was carried out for 8 days. This AS-1anti-sense DNA was able to strongly reduce the protein-argininemethyltransferase activity in U266S cells as measured by incorporationof tritiated-methyl groups to the R1 peptide substrate (FIG. 11), andwas used to investigate the role that this enzyme may play in IFNaction. The growth-inhibitory activity of IFN was chosen because it canbe most directly quantified on cells and because an interaction of ratPRMT1 with growth-related gene products has been observed (Lin et al,1996). Addition of the antisense-1 oligonucleotide AS-1, which iscomplementary to the sequence around the initiation codon of IR1B4/PRMTcDNA, reduced the growth inhibitory effect of IFN-β on human myelomaU266S cells (FIG. 12). This means that, in the presence of anti-senseAS-1, the IFN-treated cells exhibited a higher growth (excluding anytoxic effect of phosphorothioates). The growth in the absence of IFN wasnot significantly affected. The sense oligonucleotide S-3 correspondingto the same cDNA region had only a small effect (S-3, FIG. 12) ascompared to antisense-1. Sense S-3 also had only a slight inhibitoryeffect on the level of enzyme activity (FIG. 11). Another anti-sensephosphorothioate oligonucleotide AS-2 (SEQ ID NO:13), directed to themiddle of the cDNA and complementary to nucleotides 572–592 of SEQ IDNO:7, had almost no effect (FIG. 12). The up to 5 fold reduction in thegrowth inhibitory effect of IFN-β on myeloma cells, which were renderedpartially deficient in PRMT activity by antisense-1 oligonucleotidedemonstrates that the association of the IR1B4/PRMT enzyme with the ICdomain of the IFNAR1 receptor is functionally significant for IFN actionon cells.

These experiments also demonstrate that the IR1B4 protein methylatespeptide substrates of the PRMT class of enzymes, such as the R1 peptideGly-Gly-Phe-Gly-Gly-Arg-Gly-Gly-Phe-Gly (SEQ ID NO:11; Najbauer et al,1993), which was used in-the experiment illustrated in FIG. 11.Methylation of proteins on arginine residues next to glycine residues(e.g., as in the above peptide) could be a type of protein modificationwhich, like phosphorylation, serves to transduce signals into the cell.The hnRNP group of proteins is a target for PRMT enzymes, and sincethese proteins affect mRNA processing, splicing, transport and stability(Liu and Dreyfuss, 1995), their methylation may play a role inpost-transcriptional controls of gene expression. The IR1B4/PRMTprotein, discovered here as binding to a chain of the IFN receptor,could mediate changes in gene expression in response to IFN. Otherprotein substrates may become methylated through the IFN receptor,including other components of the IFN receptor complex and transcriptionfactors. Lin et al (1996) have observed that the binding of rat PRMT1 togrowth factor-induced proteins activates PRMT1 and modifies itssubstrate specificity, possibly by removal of some inhibitory proteinsassociated with PRMT1 in the cytoplasm of cells. A similar activation ofIR1B4 bound to the IFNAR1 chain of the IFN receptor can be expected.

CONCLUSIONS

A new protein IR1B1 is described which interacts with theintracytoplasmic domain of the IFNAR1 chain of the type I interferonreceptor. This protein is induced very rapidly and transiently followingIFN treatment of human cells. IR1B1 is characterized by the presence ofhelix-loop-helix EF-handle sites which are the hallmark ofcalcium-binding proteins. Calcium ion fluxes have been implicated in themechanism of action of IFNs, and in particular for the initial cellresponses and changes in cell morphology and in cytoskeletonorganization (Tamm et al, 1987). Calcium ion-activated enzymes couldproduce second messengers, such as diacyl-glycerol, in response to IFNs.Furthermore, calmodulin-like proteins regulate a number of proteinkinases and these pathways have been observed to function in IFN-treatedcells (Tamm et al, 1987). It is likely that the IFN receptor bindingprotein IR1B1 is involved in such Ca⁺⁺-dependent effects of IFNs oncells.

The two-hybrid screening for proteins interacting with the IFNAR1-ICdomain also identified another protein IR1B4, which turned out to be amember of the protein-arginine methyl transferase family of enzymes(PRMT1; Lin et al, 1996). This enzyme is known to methylate a number ofRNA and DNA binding proteins, in particular heterologous nuclearribonucleoproteins (hnRNPs). The hnRNPs are involved in mRNA transportfrom nucleus to cytoplasm, alternative splicing of pre-mRNA, andpost-transcriptional controls (Liu and Dreyfuss, 1995). The IR1B1 andIR1B4/PRMT1 proteins which dock onto the IFNAR1-IC domain reveal novelsignaling mechanisms of IFNs that exist besides the known Jak-Statpathways described by Darnell et al (1994).

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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1. An isolated recombinant DNA molecule comprising a nucleotide sequenceencoding a protein having the amino acid sequence of SEQ ID NO:2.
 2. Anisolated recombinant DNA molecule in accordance with claim 1, furthercomprising a promoter operably-linked to said nucleotide sequence suchthat said promoter promotes the transcription of said nucleotidesequence when the recombinant DNA molecule is in an appropriateexpression host.
 3. An isolated recombinant DNA molecule according toclaim 2, wherein said promoter is a constitutive promoter in humancells.
 4. An isolated recombinant DNA molecule in accordance with claim1, wherein said nucleotide sequence is SEQ ID NO:1.
 5. An expressionvector comprising a recombinant nucleic acid molecule comprising a DNAmolecule, of SEQ ID NO: 1 operably-linked in the anti-sense orientationto a promoter.
 6. An expression vector according to claim 5, whereinsaid nucleotide sequence of SEQ ID NO: 1, when in the sense orientation,includes the initiation codon at the 5′-end of SEQ ID NO:
 1. 7. Anisolated recombinant DNA molecule according to claim 2, wherein saidpromoter is an interferon-inducible promoter.
 8. An isolated recombinantDNA molecule according to claim 1, which is an expression vector.
 9. Anisolated host cell which includes an expression vector in accordancewith claim
 8. 10. A method for producing a protein having the amino acidsequence of SEQ ID NO:2, comprising culturing a host cell transfectedwith a vector containing the DNA molecule of claim 1, and isolating theprotein expressed thereby.
 11. An isolated anti-sense RNA molecule whichcorresponds to a DNA molecule comprising the nucleotide sequence of SEQID NO: 1 in the anti-sense orientation.